ANÁLISIS MOLECULAR DEL MODELO EN RATÓN DE LUPUS HUMANO

Abstract

Systemic lupus erythematosus (SLE) is a chronic multifactorial autoimmune disease, where autoantibodies cause inflammation, pain and tissue damage. Its etiology is unknown, however, multiple genetic and environmental factors, hormones and some drugs, antibiotics and infections are involved, which together exceed the threshold of immunologic tolerance. Our research group proposed that lipids associated in non-bilayer phospholipid arrangements (NPA) are involved in the development of SLE and other autoimmune diseases. These structures are found in cell membranes, and are transient, but when they become permanent they are immunogenic and induce the formation of anti-NPA autoantibodies. This proposal was confirmed by the development of a disease very similar to human SLE in BALB/c and NIH mice, by the administration of the drugs chlorpromazine (CPZ), procainamide or hydralazine that induce the stable formation of NPA on plasma membrane, or by the administration of liposomes bearing NPA induced by these drugs or by manganese. The murine model of lupus has allowed us to perform different studies to understand and follow the course of this disease, however, the genetic profile has not been identified, so in this work, we used the microarray technology for this determination. We worked with 4 groups of five female BALB/c mice. Physiological saline solution was administrated to group I, smooth liposomes without NPA to group II, liposomes with NPA induced by Mn++ or CPZ to group III and IV respectively. All groups received the corresponding solutions or liposomes by intrasplenic via twice and then intraperitoneally each week for 4 months. The mice that received liposomes with NPA induced with CPZ or Mn++ presented facial lesions with alopecia on the second and third month, respectively, after the beginning of the administration of liposomes; in both groups anti-NPA autoantibodies were detected at the end of the first month by cytofluorometry and liposomal-ELISA and anti-cardiolipin autoantibodies by ELISA at the second month, but in lower titer than anti-NPA. Mice in groups I and II did not show facial lesions or autoantibodies titers throughout the study. Mice were sacrificed on the fourth month after the beginning of the administration, because they showed high titers of both autoantibodies. Molecular analysis was done in the spleens, because this lymphoid organ contains a greater amount of mononuclear cells, which are those that have presented a greater variation in gene expression in patients with SLE. Genome-wide Agilent microarrays were made from the RNA of the spleens. By comparing the gene expression profile of groups I and II it was found that there was no variation, and the genes that are over or under expressed are not related to autoimmune diseases. By comparing the groups that developed the disease (III and IV) with control (II), we found that some of the overexpressed genes encode complement factors, and molecules involved in presentation of exogenous antigens, antibody production and TLR-4 or NOD-2 signaling, and the ones with decreased expression correspond to Natural Killer (NK) cell recognition and apoptosis. When comparing groups III and IV we found that the genes mentioned above are expressed equally in both conditions. The analysis of the gene expression profile suggests that: 1) the TLR-4 signaling via TRIF leads to a increase in interferon type 1, one of the main features of SLE, 2) the over-expression of type 1 interferon, and the decrease in NK cell recognition and apoptosis lead to an increase of autoreactive cells, which leads to a worsening of SLE, 3) over-expression of antigen presentation and antibody production also favor SLE, and 4) over-expression of complement component cell lysis support the model proposed in the study. By identifying the genetic profile of this model and comparing it with the human lupus profile, we can understand the similarity between the two diseases and gain a better understanding of the molecular and the immunological basis of autoimmune diseases, leading to the development of better strategies for their treatment.